The aim of this project is to identify and characterize HIV-infected individuals from the CAPRISA 002 cohort with broadly neutralizing antibodies that target quaternary neutralization epitopes (QNE) on the HIV envelope. To date we have identified 2 such individuals from among the 28 so far studied (Dr Morris). One of these, CAP256 developed potent antibodies against QNE that included the V1V2 region (Dr Moore). Interestingly this individual was super-infected (Dr Williamson) and viral evolution studies will be performed to indentify the second infecting virus as well as the recombinant strains to ascertain their role in the development of these antibodies. As part of this project we anticipate identifying another 9 subjects with neutralizing antibodies that target QNE from among the additional 90 that will be screened. This will be done by performing neutralization assays on a large panel of multi-subtype pseudoviruses; those with >60% neutralization breadth and who do not have anti-gp120 or anti-MPER neutralizing antibodies will be considered as having antibodies against QNE. Envelope genes from selected time-points during the development of neutralization breadth will be sequenced and functional pseudotypes tested for neutralization sensitivity to identify putative sites. We will furthermore make use of chimeric and mutant viruses to identify neutralization escape mutations as a way of identifying the antibody targets. The fine mapping and structure of these QNE epitopes will be done in collaboration with Dr Pinter (Project 1). Monoclonal antibodies will be made from those individuals where the neutralizing activity is attributable to a single specificity by Dr James Robinson (Project 4). This information will be used to design immunogens that will be tested in animal models by Dr Shiu-lok (Project 3).